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Prion biology problem space: Mad cows, itchy sheep and protein structure Touring the problem space I. II. III. IV. V. History and introduction to prion biology Idea of conformational change Exploring sequences of prion proteins Exploring physical properties and structures of prion proteins Linking evolution, structure and function of prion proteins Touring the problem space I. History and introduction to prion biology II. Idea of conformational change Exploring sequences of prion proteins Exploring physical properties and structures of prion proteins Linking evolution, structure and function of prion proteins III. IV. V. Prion History 17th ct. “Sheep are strangely dizzy” – scrapie described in Europe 1950’s: Carleton Gajdusek studied cannibalistic ceremonies of Fore tribe in New Guinea and discovered the Kuru disease Kuru attacks dura matter of brain and creates vacuoles, causing infected person to deteriorate and die within 3 months Prion History 1700s: Scrapie behavior described 1960: Alper - DNA destroying UV radiation doesn’t prevent scrapie infections. 1966: Gajdusek - Chimps get Kuru from ingesting infected brain tissue. 1967: Griffith - Speculated that TSE’s due to protein conformational changes. 1981: Mertz - Fibrils detected in scrapie infected mouse brains. (CONTINUED…) http://www.css.edu/USERS/pstein/CHM3430/11 1997: Stanley Prusiner proposed a link between kuru, CJD, scrapie and BSE. -Proposed that “prion” was responsible for spongiform manifestations in BSE-infected patients DEFINITION: PRION = Proteinaceous Infectious Particles Prion protein exists in two forms: PrPc vs. PrPsc Protein folding as we know it es.google.com/imgres?imgurl=press2.nci.nih.gov/sciencebehind/snps_cancer/snps_cancer/images/21.jpg&imgrefurl=http://press2.nci.nih.gov/sciencebehind/snps_cancer/snps_cance D%26ie%3DUTF-8%26sa%3DG Touring the problem space I. History and introduction to prion biology II. Idea of conformational change III. Exploring sequences of prion proteins Exploring physical properties and structures of prion proteins Linking evolution, structure and function of prion proteins IV. V. Prion protein folding kfolding-c kfolding-sc kunfolding-c kunfolding-sc PrPc Intermediates? PrPsc Prion folding model as link between chemistry and biology What do folding rates and equilibrium constants mean? Folded Keq Unfolded If a protein has stability of 7 kcal mol-1, how many molecules are unfolded at 37oC? Would you expect PrP to be more or less stable than other proteins? One protein: Two structures PrPc PrPsc “NORMAL” “BAD” conformation conformation http://www.uccs.edu/~rmelamed/MicroFall2002/Chapter%2010/Prion%20Structure.html Not all pretty pictures can be trusted! Based on NMR results Proposed based on lowresolution structural studies PrPc PrPc PrPc PrPsc PrPc PrPc PrPc PrPc PrPc PrPc PrPc PrPc PrPc PrPC PrPSC PrPC PrPsc fibrils Plaque Disease transmission modes Infections (Kuru, scrapie, bovine spongiform encephalophathies, etc) Inherited (Creutzfeld-Jacob disease, Gerstmann-Straussler-Scheinker disease, Fatal familial insomnia) Sporadic (BSE, CJD ?) Link to The Case of the Cherry Hill Cluster By D.T. MAX NY Times magazinePublished: March 28, 2004 Prion diseases and Koch’s postulates I. II. III. IV. The agent must be present in every case of the disease The agent must be isolated from the host and grown in a lab dish The disease must be reproduced when a pure culture of the agent is inoculated into a healthy susceptible host The same agent must be recovered again from the experimentally infected host. Can this be applied to understanding prion diseases? Touring the problem space I. II. History and introduction to prion biology Idea of conformational change III. Exploring sequences of prion proteins IV. Exploring physical properties and structures of prion proteins Linking evolution, structure and function of prion proteins V. Global view of the amino acid sequence of PrP Multiple ways to visualize and analyze protein sequence Use GeneDoc from Pittsburgh Supercomputing Center to analyze multiple prion sequences By using different coloring schemes, can visualize and quantify conservation of different properties Comparison of human, cow, sheep and mouse prion protein sequence Conservarion of polar vs. non-polar pattern PROPERTIES Level 1 DEHKRNQT LIVMFYWAGCP POLAR NON-POLAR Analyzing charge distribution Level 2 DEHKR NQST LIVMFYW AG CHARGED POLAR UNCHARGED HYDROPHOBIC SMALL hydrophobic Expanded amino acid properties Examples of questions that can be examined through sequence analyses Determining evolutionary relationships among the various organisms examined. Investigating how amino acid sequence may be linked to the overall structure of the protein Examining the role of repetitive elements in prion homologies. Touring the problem space I. II. III. History and introduction to prion biology Idea of conformational change Exploring sequences of prion proteins IV. Exploring physical properties and structures of prion proteins V. Linking evolution, structure and function of prion proteins Exploring physical characteristics How big is the prion protein? How does that compare to other proteins? Where is it localized in the cell? Is its size or shape unusual? Is the distribution of amino acids unusual? PROTPARAM: a tool for analysis of physical properties http://ca.expasy.org/cgi-bin/protparam Number of amino acids: 253 Molecular weight: 27629.1 Theoretical pI: 9.23 Amino acid composition: Ala (A) 10 4.0% Arg (R) 11 4.3% Asn (N) 12 4.7% Asp (D) 6 2.4% Cys (C) 4 1.6% Gln (Q) 14 5.5% Glu (E) 9 3.6% Gly (G) 45 17.8% His (H) 10 4.0% Ile (I) 9 3.6% Leu (L) 12 4.7% Lys (K) 11 4.3% Met (M) 11 4.3% Phe (F) 7 2.8% Pro (P) 17 6.7% Ser (S) 15 5.9% Thr (T) 13 5.1% Trp (W) 9 3.6% Tyr (Y) 13 5.1% Val (V) 15 5.9% Visualizing protein structure with Cn3D Secondary structural elements can be analyzed in isolation Helices can be dissected by mapping onto helical wheel http://bioinf.man.ac.uk/%7Egibson/HelixDraw/helixdraw.html Examples of questions that can be explored through structural analyses Some mutations have been associated with prion diseases. By mapping these onto structures, propose a hypothesis to explain this. Most of the variation seems to be in the Nterminal region. Is there homology to this region in other proteins? Touring the problem space I. II. III. IV. V. History and introduction to prion biology Idea of conformational change Exploring sequences of prion proteins Exploring physical properties and structures of prion proteins Linking evolution, structure and function of prion proteins What does PrP do? •GPI- anchored cell surface protein •Knockout mice mostly normal •Might be involved in some signaling cascades •Interaction with hypothetical “protein x” might be needed for structural conversion What makes prion a prion? Why don’t other proteins adopt two drastically different stably folded conformations? A helix is a helix…but why are helices in prion protein likely to undergo a big structural change? How would you test/further explore these questions? “Big picture” questions as a platform for development of testable hypotheses Why would nature evolve prions? How would you tell a prion if you saw one? Yeast prions do not cause a disease. Are there other non-harmful prion-like proteins? Can prions be beneficial?